Neisseria gonorrhoeae causes the sexually-transmitted disease gonorrhea as well as pelvic inflammatory disease, sepsis, meningitis, and arthritis. Gonococci generate an incredible degree of genetic diversity and antigenic diversity allowing them to avoid the human immune response. A significant amount of the genetic diversity is created through natural transformation. Natural transformation is a way of life for gonococci. They are constitutively competent for transformation, and they preferentially take up DNA from other gonococci or other Neisseria, i.e., they take up DNA that is most likely to be useful for recombination with the genome. We discovered a type IV secretion system (T4SS) encoded in a genomic island in the gonococcal chromosome. The T4SS secretes chromosomal DNA from the bacteria into their surroundings. The secreted DNA is active in the transformation of other gonococci in the vicinity. We demonstrated that the secreted DNA is single- stranded, blocked at the 5' end, and that for it to be secreted it must first be processed by a relaxase that acts at a single site in the chromosome, the origin of transfer. The T4SS has effects on host cell interactions. It also affects adherence of the bacteria to each other and to cells and surfaces. This proposal is focused on how expression of the T4SS is regulated. As is the case for many other T4SSs, the gonococcal type IV secretion genes are repressed under normal culture conditions. Many T4SS proteins are not detectable by western blot and immune-gold electron microscopy identifies a single spot on a subpopulation of cells. These results suggest that each cell might have one T4SS apparatus or that only a few cells in the culture produce the secretion system. To further study the mechanism of secretion, to identify additional secretion substrates, and to elucidate the effects of the type IV secretion during infection, the mechanism controlling type IV secretion will be determined. The specific aims of this proposal are: 1) to identify regulators and environmental conditions that affect T4SS expression, 2) to determine the mechanisms and effects of the traH regulatory region on T4SS transcription, translation, and protein localization, and 3) to use model substrates and whole genome sequencing to examine the mechanism and consequences of DNA secretion.